Dengue virus infections are a serious cause of morbidity and mortality in many areas of the world. Most infections cause uncomplicated dengue fever (D), but severe illness, dengue hemorrhagic fever (DHF) also occurs. DHF is much more commonly observed during secondary infections with a different serotype of dengue virus from that which caused the primary infection. It is assumed that DHF is caused by immunopathological mechanisms. In this Program Project, we will define immunopathological mechanisms which may lead to DHF in order to improve treatment of DHF and provide a basis for the development of safe and effective vaccines. This goal of this Basic Immunology Research Core is to develop and modify molecular, immunological and virological assays to We will: 1) develop PCR techniques in order to detect mRNA for cytokines to determine TcR Valpha and Vbeta gene usage, and to detect dengue virus genome. 2) develop PCR-based limiting dilution assays in order to quantitate cells which contain cytokine mRNA and to quantitate dengue virus-infected cells 3) develop in situ PCR technique in order to determine cells which contain cytokine mRNA and dengue virus-infected cells 4) develop recombinant vaccinia viruses which contain genes coding for dengue virus proteins to be used in CTL assays The development of these assays and reagents by this Core is essential for the success of Projects 1 and 2, and Clinical Research Laboratory Core. The assays developed in this Core will be transferred to the Core in Thailand and to the laboratories at the University of Massachusetts where project 2 is performed.